Devices, systems and methods for high-throughput electrophysiology

ABSTRACT

Electrophysiology culture plates are provided and are formed from a transparent micro-electrode array (MEA) plate. The MEA plate comprises a substrate, a first layer and a first insulating layer. The substrate has a plurality of vias extending from an upper to a lower surface, each via being in electrical contact with each of a plurality of contact pads disposed on the lower surface. The first layer is disposed on the upper surface of the substrate and has a plurality of MEA arrays in in electrical communication with at least a first routing layer. Each MEA array comprises a plurality of reference electrodes and a plurality of microelectrodes and the first routing layer is in electrical communication with a select number of the plurality of vias. A first insulating layer is disposed on the first layer. The MEA plate is joined to a biologic culture plate having a plurality of culture wells such that each culture well defines an interior cavity having a bottom surface that is at least partially transparent and in positioned in registration with a select optical port. The MEA plate is coupled to the biologic culture well plate such that each MEA array is operatively coupled to one culture well wherein each microelectrode and each reference electrode are in electrical communication with the interior cavity through the bottom surface of the culture well.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. application Ser. No.14/279,961, filed May 16, 2014, and U.S. Provisional Application No.61/858,945, filed Jul. 26, 2013, each of which is hereby incorporatedherein by reference in its entirety.

BACKGROUND

1. The Field of the Invention

Implementations described herein relate generally to high-throughputelectrophysiology culture system and, more particularly, to anelectrophysiology culture system with a culture plate having anintegrated monolithic microelectrode array plate.

2. Related Art

In vitro electrophysiology culture systems having microelectrode arrays(MEAs) can provide important insights into networks of electricallyactive cells. MEA-based electrophysiology culture systems can beconfigured to concurrently monitor single cell and network levelactivity over extended periods of time and without affecting the cellculture under investigation. Since their instrumental role in the 1993landmark discovery of spontaneous waves in a developing retina, thevariety and scope of MEA-based electrophysiology applications havedramatically expanded. Recently, for example, MEA-basedelectrophysiology culture systems have been used to investigate thesuppression of epileptic activity and in the study of novel plasticitymechanisms in cultured neural networks. Advances in cell culturepreparations have similarly led to applications for MEA-basedelectrophysiology culture systems in the fields of drug screening,safety pharmacology, and biosensing.

Present day MEA-based electrophysiology culture systems are typicallydesigned around small-footprint, single-well devices. However, thecomplete analysis of complex cellular systems and processes can requirerepeated experiments. The number of experiments can increase quicklywhen considering multiple variables, such as stimulus size, compoundtype, dosage strength and the like. Thus, the small-scale format oftraditional MEA systems presents problems due to excessive experimentaland statistical sizes, whereby the serial nature of these devices canrender even basic investigations time and cost prohibitive. As oneillustrative example, a researcher examining the effect of pythrethroidson two-hour spontaneous activity recordings can require 8 doses ofpermethrin, with an N of 6 for each dose. With traditional MEA-basedelectrophysiology culture systems, this very simple experiment canrequire over $5,000 in MEA-based electrophysiology culture plates (or“MEA culture plate”) and 50 to 60 man-hours. The time investment canfurther increase with the logistics of culturing, maintaining, andtesting dozens of individual specimen.

Thus, design of a high-throughput MEA culture plate is highly desirable.However, conventional manufacturing techniques fall short of enablingtheir manufacture by merely scaling up a conventional design. Forhigh-throughput investigations, large-area, American National StandardsInstitute (ANSI)/Society for Lab Automation and Screening(SLAS)-compliant plates can be important as industry standard compliancecan provide compatibility with other high-throughput instrumentationsuch as plate readers and robotics handlers. Conventional MEA cultureplates, some of which can be subdivided into a small number of wells(e.g., about 6), can cost from about $150 to about $500 and are noteasily scaled to high well count plates without prohibitivemanufacturing costs. Specifically, the development of anANSI/SLAS-compliant, high-throughput MEA culture plate presents twomajor challenges: (1) ensuring 100% yield of widely distributedmicro-scale electrodes and (2) developing cost-effective manufacturingprocesses to provide inexpensive high-throughput MEA culture plates. Themicrofabrication industry has traditionally addressed these issues byfabricating thousands of micro-scale devices in parallel and thenindividualizing each unit, with the results of reducing the per-unitcost of each device and ensuring that non-working units can quickly beidentified and discarded. The working units are then packaged usingwafer-level packaging technologies or individual unit-level technologiesthat have been optimized for cost-effectiveness. However, forhigh-throughput MEA plate fabrication, the plate size is much greaterthan traditional micro-scale devices, increasing the likelihood that asingle microelectrode may fail, rendering the entire plate a non-workingunit. Additionally, if only single plate can be microfabricated on onewafer, the cost advantage is lost versus the batch fabrication ofmicro-scale-sized devices described above.

Thus, broad access to neural information along with the minimallyinvasive nature of a MEA-based electrophysiology culture systems rendersthem a potentially valuable tool for discovery. However, the throughputof MEA-based electrophysiology culture systems needs to increase to keeppace with the requirements of today's researchers. Accordingly, a needexists for improved MEA-based electrophysiology culture systems thatprovide for high-throughput applications and reliable large-areamicrofabrication methods to manufacture the MEA plates and, ultimately,the assembled MEA-based electrophysiology culture plates.

SUMMARY

It is to be understood that this summary is not an extensive overview ofthe disclosure. This summary is exemplary and not restrictive, and it isintended to neither identify key or critical elements of the disclosurenor delineate the scope thereof. The sole purpose of this summary is toexplain and exemplify certain concepts of the disclosure as anintroduction to the following complete and extensive detaileddescription.

In one aspect, the present disclosure describes an electrophysiologyculture plate having an ANSI or SLAS-compliant format comprising abiologic culture plate coupled to a monolithic MEA plate. A monolithicMEA plate can have a plurality of layers having electrodes, electroderouting and vias that ultimately provide for electrical communicationbetween the culture well contents and an electronics unit.

In another aspect, the present disclosure provides for anelectrophysiology culture system comprising an electrophysiology cultureplate and an electronics unit configured to stimulate at least one celland immediately record the response after stimulation.

Additional features and advantages of exemplary implementations of theinvention will be set forth in the description which follows, and inpart will be obvious from the description, or may be learned by thepractice of such exemplary implementations. The features and advantagesof such implementations may be realized and obtained by means of theinstruments and combinations particularly pointed out in the appendedclaims. These and other features will become more fully apparent fromthe following description and appended claims, or may be learned by thepractice of such exemplary implementations as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate aspects and together with thedescription, serve to explain the principles of the methods and systems.

FIG. 1 is a schematic drawing depicting one implementation of a highthroughput electrophysiology culture system of the present disclosure.

FIG. 2 depicts an exploded perspective view of one implementation of anelectrophysiology culture plate of the present disclosure.

FIG. 3 depicts one exemplary implementation of a first layer of themonolithic microelectrode array plate.

FIG. 4 depicts one exemplary implementation of a second layer of themonolithic microelectrode array plate.

FIG. 5 depicts one exemplary implementation of a third layer of themonolithic microelectrode array plate.

FIG. 6 depicts one exemplary implementation of a fourth layer of themonolithic microelectrode array plate.

FIG. 7 depicts the first layer, second layer, third layer and fourthlayers of the monolithic microelectrode array plate superimposed uponeach other.

FIG. 8 depicts the underlying portion of a monolithic MEA plate in oneimplementation of a culture well plate having 48 wells.

FIG. 9 illustrates one exemplary process flow schematic for defining theelectrical routing and contact pats on one implementation of a PCBsubstrate.

FIG. 10 illustrates one exemplary process flow schematic for definingthe electrodes.

FIG. 11A shows an optical micrograph of one implementation of ananotextured gold electrode. FIG. 11B shows an SEM micrograph of oneimplementation of a nanotextured gold electrode.

FIG. 12 illustrates one exemplary process flow for monolithicmicroelectrode array fabrication on a glass substrate.

FIG. 13A shows a perspective view of one aspect of an optical micrographof an assembled 48-well MEA fabricated on an opaque panel. FIG. 13Bshows an enlarged view of the optical micrograph of the assembled48-well of FIG. 13A.

FIG. 14 shows exemplary impedance spectroscopy measurements for onerepresentative set of nanotextured microelectrodes.

FIG. 15 is a graph illustrating the RMS noise of a single well in anexemplary electrophysiology culture plate of the present disclosure.

FIG. 16 depicts the cytotoxicity results of testing an electrophysiologyculture plate of the present disclosure against a conventionalpolystyrene culture plate.

FIGS. 17A shows a perspective top view of one aspect of an MEA culturewell plate having alignment features, keying features and features toaccommodate an IC chip. FIG. 17B shows a perspective bottom view of theMEA culture well plate of FIG. 17A.

FIG. 18 shows an enlarged view of the top of MEA culture well plate ofFIG. 17A.

FIG. 19 shows an enlarged view of the bottom of the MEA culture wellplate of FIG. 17A.

FIG. 20A is a cross sectional view of one aspect of a culture wellhaving a smaller footprint at the bottom of the well as compared to thetop of the well. FIG. 20B is a perspective view of the culture wellshown in FIG. 20A.

FIG. 21 shows a perspective view of one aspect of a culture well platelid.

FIG. 22 depicts one aspect of a clamping mechanism for clamping a MEAculture well plate.

FIG. 23 is an enlarged view of the clamping mechanism depicted in FIG.22.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description, examples, drawing, and claims, and theirprevious and following description. However, before the present devices,systems, and/or methods are disclosed and described, it is to beunderstood that this invention is not limited to the specific devices,systems, and/or methods disclosed unless otherwise specified, as suchcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting.

The following description of the invention is provided as an enablingteaching of the invention in its best, currently known aspect. To thisend, those skilled in the relevant art will recognize and appreciatethat many changes can be made to the various aspects of the inventiondescribed herein, while still obtaining the beneficial results describedherein. It will also be apparent that some of the desired benefitsdescribed herein can be obtained by selecting some of the featuresdescribed herein without utilizing other features. Accordingly, thosewho work in the art will recognize that many modifications andadaptations to the present invention are possible and can even bedesirable in certain circumstances and are a part described herein.Thus, the following description is provided as illustrative of theprinciples described herein and not in limitation thereof.

Reference will be made to the drawings to describe various aspects ofone or more implementations of the invention. It is to be understoodthat the drawings are diagrammatic and schematic representations of oneor more implementations, and are not limiting of the present disclosure.Moreover, while various drawings are provided at a scale that isconsidered functional for one or more implementations, the drawings arenot necessarily drawn to scale for all contemplated implementations. Thedrawings thus represent an exemplary scale, but no inference should bedrawn from the drawings as to any required scale.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding described herein. It will beobvious, however, to one skilled in the art that the present disclosuremay be practiced without these specific details. In other instances,well-known aspects of electrophysiology culture systems andmicroelectromechanical systems (MEMS) have not been described inparticular detail in order to avoid unnecessarily obscuring aspects ofthe disclosed implementations.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Ranges may be expressed herein as from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another aspect includes from the one particularvalue and/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another aspect. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal aspect. “Such as” is not used in arestrictive sense, but for explanatory purposes.

Disclosed are components that can be used to perform the disclosedmethods and systems. These and other components are disclosed herein,and it is understood that when combinations, subsets, interactions,groups, etc. of these components are disclosed that while specificreference of each various individual and collective combinations andpermutation of these may not be explicitly disclosed, each isspecifically contemplated and described herein, for all methods andsystems. This applies to all aspects of this application including, butnot limited to, steps in disclosed methods. Thus, if there are a varietyof additional steps that can be predefined it is understood that each ofthese additional steps can be predefined with any specific aspect orcombination of aspects of the disclosed methods.

Implementations described herein are directed toward devices, systemsand methods for high-throughput electrophysiology. More particularly,the present disclosure is directed to an electrophysiology culturesystem with a culture plate having an integrated monolithicmicroelectrode array plate. For example, one or more implementationsdescribed herein provide for an electrophysiology culture platecomprising a monolithic MEA plate having a plurality of MEAs and abiologic culture plate having a plurality of culture wells wherein theMEA plate underlies and is coupled to the culture well plate such thateach MEA is operatively coupled to one culture well of the plurality ofculture wells. In other aspects, the present disclosure is directed toan electronics unit configured to receive the electrophysiology cultureplate and, in yet other aspects, to mechanical features provided on boththe electrophysiology culture plate and the electronics unit tofacilitate placement and advantageous operational modalities of theelectrophysiology culture system.

Reference will now be made to the drawings to describe various aspectsof one or more implementations of the invention. It is to be understoodthat the drawings are diagrammatic and schematic representations of oneor more implementations, and are not limiting of the present disclosure.Moreover, while various drawings are provided at a scale that isconsidered functional for one or more implementations, the drawings arenot necessarily drawn to scale for all contemplated implementations. Thedrawings thus represent an exemplary scale, but no inference should bedrawn from the drawings as to any required scale.

High-throughput screening (HTS) tools make use of multiwell biologicculture plates that follow exacting guidelines established by theSociety for Lab Automation and Screening (SLAS) and the AmericanNational Standards Institute (ANSI). These standards are adhered to byall HTS supporting equipment such as, for example and withoutlimitation, plate readers, robotic handlers, liquid handling devices andthe like. Compliance with these standards can enable a high-throughputMEA platform to achieve full potential, as it leverages existinghigh-throughput infrastructure including the automation of mediaexchanges and compound delivery. Adherence to the standard, however,requires defining micro-scale structures across a single, large-areaplate, which, in turn, can dramatically escalate costs.

Implementations disclosed herein comprise an electrophysiology cultureplate assembly and electronics unit docking design based on a verticallyintegrated footprint constrained by the outer dimensions of theANSI/SLAS microtiter plate format. The high-throughput electrophysiologyculture plate can comprise a multiwall biologic culture plate integratedwith an MEA plate. In one aspect, cost-effective, scalable technologiesto address these problems are disclosed. In a further aspect, 1-, 6-,12-, 24-, 48-, 96- and other standard well configuration MEAs on asingle monolithic substrate along with their manufacture are disclosed.In some aspects, innovations in inexpensive, standard technologies suchas injection molding, die cutting and laser cutting are leveraged toenable modular assembly of these electrophysiology culture well plates.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding described herein. It will beobvious, however, to one skilled in the art that the present disclosuremay be practiced without these specific details. In other instances,well-known aspects of electrophysiology culture systems andmicroelectromechanical systems have not been described in particulardetail in order to avoid unnecessarily obscuring aspects of thedisclosed implementations.

Turning now to FIG. 1, an implementation of one exemplary aspect of ahigh-throughput electrophysiology culture system 100 is illustrated. Anelectrophysiology culture plate 102 can comprise a monolithic MEA plate104 integrated with a biologic culture plate 106, and electronics 108together with software configured to stimulate a cell culture via theelectrophysiology culture plate to evoke a response and to record data.The electrophysiology culture plate can comprise a plurality of culturewells configured to culture electroactive cells. A grid of tightlyspaced microelectrodes 112 configured to extracellularly interface withthe cultured cells is operatively associated with each culture well 114.Each electrode can be configured to record electrical activity fromnearby neurons and electrically stimulate those cells. This techniquecan provide an extracellular, label free method for examining bothindividual neuronal behavior and overall network activity, optionally,simultaneously. Mechanical features can also be provided that operate tocouple the MEA culture well plate to the electronic unit. In a furtheraspect, these mechanical features can be configured so as not tointerfere with topside access to the electrophysiology culture plate. Inanother aspect, the electronics unit can be used amplify and filter thelow amplitude extracellular signals captured by both the microelectrodesand reference electrodes and, in other aspects, provide user directedstimulation to the cells. In further aspects, the electronics unit canconvert the analog electrode signals received from the cells into datathat can be used and manipulated by the computer software, whileminimizing the amount of noise injected into the very low amplitudesignals that are measured (i.e., extracellular recordings).

For high-throughput electrophysiology, large-area, ANSI/SLAS-compliantmicro-electrode array (MEA) plates can be important as industry standardcompliance can provide compatibility with other high-throughputinstrumentation such as, for example and without limitation, platereaders, robotics handlers and the like. However, distributinghundreds-to-thousands of micro-scale sensors across a macro-area platecan present challenges within modern microfabrication and process/devicedesign. The present disclosure describes devices, systems and methods toform an MEA-based electrophysiology culture plate having the followingcharacteristics important to achieving an ANSI/SLAS-compliant,high-throughput MEA:

In one aspect shown in FIG. 2, the present disclosure can provide forhigh-throughput MEA culture well plates 102 that comprise an MEA plate104 that can be fully vertically integrated with a culture well plate106. In a further aspect, the monolithic MEA plate 104 and culture wellplate 106 can be joined by an intermediate adhesive 116. In anotheraspect, the monolithic MEA plate can be monolithic, and, in additionalor alternative aspects, can comprise, for example and withoutlimitation, polymers, glass, glass-reinforced epoxy resin and the like.

MEA Plate

One aspect of the present invention shown in FIGS. 3-8 comprises amonolithic MEA plate having a plurality of layers having electrodes,electrode routing and vias that ultimately provide for electricalcommunication between the culture well contents and the electronicsunit. In one illustrative example, the monolithic MEA plate can comprisefour layers where the first layer 300 comprises a plurality of MEAarrays 302 and a first microelectrode routing 304 patterned thereon,wherein each MEA array 302 comprises a plurality of reference electrodes802 and a plurality of microelectrodes 804, a second layer 400comprising reference electrode routing 402 in electrical contact withthe plurality of reference electrodes 802, a third layer 500 comprisinga ground plane 502 in electrical contact with the reference electroderouting 402, and a fourth layer 600 comprising a second microelectroderouting 602 in electrical contact with the first microelectrode routing304 and a corresponding contact pad 604 formed on a lower major surfaceof the monolithic MEA plate. FIG. 7 illustrates the first layer 300, thesecond layer 400, the third layer 500 and the fourth layer 600superimposed on each other. FIG. 8 depicts the underlying portion of amonolithic MEA plate in one implementation of a culture well platehaving 48 wells.

In one aspect, the present disclosure can provide for bottom-sidecontact pads 504 within the footprint of the ANSI or SLAS prescribeddimensions of the MEA culture plate. In order to interface withindustry-standard robotic handlers and instrumentation, the MEA cultureplate can be configured such that no part of the contact pads extendsbeyond the ANSI or SLAS-prescribed footprint. Moreover, the contact padscan be located on the bottom of the MEA culture plate, so as toeffectively preserve the topside for experimental preparation andexecution.

In another aspect, the present disclosure can provide for high-densityelectrical contact pads on the periphery of the device. In specificembodiments where a multiplexing circuit cannot be embedded into theplate, high-density, bottom-side contact 504 pads can be positioned onthe periphery of the device. Avoiding placing contact pads in theinterior spaces facilitates optical transparency under the wells and/orthe use of multiple well configurations with the same pad configuration.

Microfabrication of Opaque HTMEAs

In one aspect, one method to form an opaque monolithic MEA plateinvolves patterning approximately 50 μm metal traces and approximately40 μm laser-patterned vias (or openings) on traditional PCB substratessuch as, for example and without limitation, FR-4, polyimide, and liquidcrystal polymer (LCP), and the like. Other polymer substrates such as,for example and without limitation, polyethylene naphthalate (PEN),polyethylene terephthalate (PET), other varieties of polyimides such astransparent polyimide, polyesters, polytetrafluoroethylene (PTFE) andthe like can optionally be substituted for polyimides, e.g., Kapton. Inan alternate or additional aspect, higher-end PCB technologies canenable much smaller features, e.g., lower than about 50 μm metal tracesand about 40 μm laser-patterned vias.

In one aspect, in order to accommodate 768 stimulation/recordingmicroelectrodes with additional integrated reference electrodes in ahigh-throughput ANSI/SLAS format, four layers of routing can be requiredto route all signals from the bottom side to the topside formicroelectrode access. Contact pads for electronics access can bedefined along the perimeter of the device. The definition of four rowsof such pads ensures the ability to connect to all 768 electrodes on thedevice in a variety of configurations without affecting ANSI/SLASstandard micro-titer culture well locations (for 1, 6, 12, 24, 48, 96,192 384, and higher well count culture well designs). The pads can beabout 1000 μm by about 800 μm and can have a pitch of about 1200 μm. Theshortest distance between the pads, however, can be about 320 μm. Theentire metal track routing in this design can be performed with about 3mil (or about 75 μm) metal track width with about 3 mil spacing in themicroelectrode area (illustrated in FIG. 8) and about 5 mil (or about125 μm) metal track width with about 5 mil spacing elsewhere, therebyensuring cost-effective production.

In one aspect of the present disclosure, the five basic PCB processesused to form the MEA plate can comprise a combination of photoengraving,milling, etching, plating and lamination. In light of the presentdisclosure, one skilled in the art will appreciate that PCB processesare typically performed on large area substrates (e.g., panels that canbe 19-inch by 14-inch or larger) and outside the cleanroom. Accordingly,PCB processes tend to be more cost-effective thanmicrofabrication/microelectronic processes, however they produce largerfeature sizes as compared to microfabrication processes. With therepetition of the five basic processes above, complex devices can becreated both on flexible and flex-rigid substrates.

In one aspect of the present disclosure shown in FIG. 9, photoengravingcan be used to define the routing traces and contact pads on a flexibleor a flex-rigid substrate. Typical PCB substrates such as, for exampleand without limitation, FR-4, Kapton, LCP and the like, can have copperlayers of various thicknesses (specified typically by weight of copperin ounces) glued or electroplated on them. Subsequently, a dry film maskcan be coated on the copper-plated PCB and imaging can be performedusing a UV lamp to define the routing features. Then, the dry filmresist can be developed and the board can be etched to remove copperfrom unwanted areas. One skilled in the art will appreciate that thecopper layer can be adhered to both sides of the PCB substrate anddouble-side processing can be performed so that both sides can beelectrically connected using the other standard processes discussedherein.

In another aspect of the present invention, milling, drilling and thelike can be used to create vias or through-holes using standardmechanical drill bits for larger vias or laser machining for smallersized vias. One skilled in the art will appreciate that laser machinedvias have the added advantage of highly controllable drill depth.

In another aspect of the present invention, both electroless plating andelectroplating of copper or other metals can be performed in standardPCB processing. Electroless plating can be used to provide a seed layeror adhesion layer for subsequent electroplating. For example and withoutlimitation, nickel can be used to create a seed layer havingnanometer-scale thickness. In another aspect, electrical contact betweenthe routing layers of the PCB can be provided through electroplatedcopper vias.

In light of the present disclosure, one skilled in the art willappreciate that, by utilizing a sequence of the five basic PCBprocedures, four routing layers can be fabricated and combined togetherinto a monolithic device using intermediate insulating layers throughlamination processes.

In another aspect, before the top metal layer can be laminated, a softgold layer can be electrodeposited on the copper. In certain aspects,the gold layer electroplating can be important in the definition of thefinal microelectrodes. In a further aspect, the soft gold layer can bedefined to achieve sufficient thickness, e.g., about 20 to about 30micro-inches or about 0.5 to about 0.762 μm) to withstand lasermicromachining that is performed at a later stage. In one example, asufficient thickness can be from about 20 to about 30 micro-inches. Inlight of the present disclosure, one skilled in the art will appreciatethat such thicknesses of electroplated metal on thin film metal can besufficient to withstand the removal of polymer materials deposited ontop of the electroplated layer utilizing laser micromachining In afurther aspect, following gold plating, a layer of Kapton can belaminated on top of the finished 4-layer rigid FR-4 with all themicroelectrodes defined and routed. In one example, the Kapton layer canbe 12.5 μm thick with 12.5 μm acrylic adhesive.

Subsequently, in another aspect depicted in FIG. 10, the Kapton can beselectively laser micromachined utilizing either a CO₂ or a UV laser todefine the microelectrodes. One skilled in the art will appreciate thatan excimer laser system operating at about 248 nm can be well suited formicromachining polymers. Laser/material interactions during ablation canbe complex and depend both on the laser characteristics such as, forexample and without limitation, wavelength, pulse duration, intensityand material characteristics such as, for example and withoutlimitation, absorption spectra, ease of evaporation and the like. In afurther aspect, the amount of material removed can be related to athreshold defined by the properties of the material being micromachined.In yet another aspect, laser micromachining processes can deposit carbonresidue on the parts, therefore plasma and solvent cleaning processescan be used to remove this residue and to complete a monolithic 48-wellopaque MEA board. FIG. 13 illustrates an optical micrograph of anassembled 48-well MEA fabricated on an opaque panel.

As shown in FIGS. 11A and 11B, it is another aspect of the presentdisclosure that the electrode characteristics can be defined by softgold electroplating and laser micromachining processes. Both of theseprocesses can define a gold layer that can be textured on a nano-scale,e.g., 100s of nanometers. In light of the present disclosure, oneskilled in the art will appreciate that texturing leads to an increasedsurface area and, thus to superior impedance performance compared tothin film process-based MEA plates.

Microfabrication of Transparent HTMEAs

Traditional microfabrication can be typically performed on substratessuch as silicon and glass. MEA plates for in vitro and in vivoapplications have been demonstrated on these substrates since the late1960s. More recently such MEA plates have been developed on a variety ofpolymers such as, for example and without limitation, parylene, Kapton,poly dimethyl siloxane (PDMS), SU-8, poly methyl methacrylate (PMMA),polyurethanes (PU) and the like. One skilled in the art will appreciatethat microfabrication affords advantages such as, for example andwithout limitation, scalability, nanoscale feature sizes, robustmanufacturability, CMOS integration (on silicon substrates only),advanced processing tool set, yield optimization for high-volumeproduction and the like. However, as discussed previously, MEA platescan require relatively large-area compatible definition of micro- andnano-scale features and hence some of the traditional advantages ofwafer-based microfabrication processes can be lost. Additionally siliconcannot be used due to transparency requirements. One skilled in the artwill appreciate that glass panel microfabrication, made popular andcost-effective by the display, flexible electronics and solar cellsindustries, is particularly well suited for the manufacture of MEAplates.

In another aspect of the invention shown in FIG. 12, the presentinvention provides for a process flow for MEA plate fabrication on aglass panel. In light of the present disclosure, one skilled in the artwill appreciate that, with minor modifications in processing steps theseglass panels can be substituted by polymer panels. Polymers such as, forexample and without limitation, polycarbonate (PC), polystyrene (PS),poly methyl methacrylate (PMMA), cyclic olefin co-polymers (COCs) andthe like can be ideal candidates for such a process but carefulconsideration needs to be paid to the processing temperatures,mechanical (e.g., roughness) and optical (e.g., transparency) propertiesof such polymers before they can be applied to transparent HTMEAs.

In one aspect of the present disclosure, vias can be created in theglass panel. In an illustrative example, the thickness of the glass canbe about 1 mm. Established technologies such as, for example and withoutlimitation, powder blasting, high precision CNC milling and lasermicromachining can be used to create the vias on the periphery of thesubstrate.

In a further aspect, the microfluidic vias and channels in a glasssubstrate can be formed by powder blasting. One skilled in the art willappreciate that powder blasting can be a flexible, cost-effective andaccurate in the present application. Powder blasting utilizesphotolithography to define the location of the vias and, subsequently,the exposed glass panel areas can be subjected to a powder that etchesglass in those locations. The photoresist-covered areas deflect thepowder such that there can be no etching in these areas. Feature sizeaccuracy of approximately 25 μm can be achieved with powder blastingwith minimal roughness (less than 2.5 μm) imparted to the vias.

In another aspect, high precision CNC milling can be utilized tomicrofabricate vias on glass panels. CNC milling can accurately machineglass over large areas. In one illustrative example, CNC milling canachieve about 5 μm placement accuracy over relatively large areas suchas, for example and without limitation, several hundred millimeters.

In another aspect, vias on glass substrates can be created using lasermicromachining One skilled in the art will appreciate that materialsused to fabricate the MEA plate will affect the type of laser bestsuited for this function. As one illustrative example, a CO₂ laser issuitable for micromachining certain varieties of glass, such as, forexample and without limitation, fused silica and the like.

In another aspect, the vias previously formed can be subjected toelectroplating, screenprinting or the like in order to establish aconductive path through the via. In light of the present disclosure, oneskilled in the art will appreciate that screenprinting can beparticularly well suited to form the vias since it can be large areaprocessing compatible technique and has the ability to produce finefeatures with excellent accuracy. Screenprinting comprises a process ofutilizing a highly intricate mesh through which a conductive adhesivecan be deposited in a predetermined pattern defined on the mesh. In oneaspect, the level of intricacy of the mesh can depend on the size of thevias or traces to be defined. Subsequently, conductive epoxy depositioncan be performed utilizing a squeegee which makes intimate contact withthe screen and whose motion can be accurately controlled. Parameterssuch as the force and speed can be optimized to produce the pattern ofthe filled conductive vias. Additionally vacuum might be required toensure complete filling of the tallest vias. In one exemplary aspect,the tallest vias can be about 1 mm tall.

In another aspect, the screen printed substrate can be polished toachieve a substrate with minimal surface roughness for furtherprocessing.

In further aspects, the screen printed glass panel can undergoadditional processing that can involve structuring the metal tracks,insulation and microelectrodes. One skilled in the art can appreciatethat standard microfabrication technologies which are well establishedin mass micro-manufacturing such as, for example and without limitation,lift-off and deposit/etch can be utilized to define the metal tracks onthe screen printed glass panel.

In other aspects, regions of insulation can be selectively defined onthe current form of the glass panel. In one illustrative example, SU-8insulation can be defined using a photolithography process. In anotherillustrative example, silicon dioxide or silicon nitride insulation canbe defined utilizing a PECVD process, followed by a photolithographyprocess and an etch process. Here, the etch process (i.e., wet or dryetch or a combination of both) can define the recordingsites/microelectrodes while the photoresist protects the rest of thedevice from etching. In yet another example, parylene can also bedefined utilizing the process described for silicon dioxide and siliconnitride. The deposition of parylene however can be a room temperaturevapor deposition.

In another aspect, microelectrodes can be formed on individualizeddevices obtained by dicing the glass panel into individual unitsutilizing a large area finesse electroplating process at the assembleddevice level. In one further aspect, the microelectrodes can be formedfrom nano-porous platinum.

Culture Plate

In one aspect of the present disclosure, a multi-well biologic cultureplate configured to be joined to the MEA plate can be provided. Inanother aspect, the biologic culture plate can further comprise a lid.The biologic culture plate and lid can be formed, for example andwithout limitation, by conventional injection molding techniques. In afurther aspect, the biologic culture plate and lid can comprisematerials such as, for example and without limitation, polystyrene,polycarbonate and the like.

In another aspect, the biologic culture plate is configured to beANSI/SLAS compliant.

In another aspect, the lid comprises a double baffled edge, to reducethe amount of fluid lost through evaporation and/or maintain sterility.

In another aspect, at least one electronics pocket can be formed on thebiologic culture plate to allow space for IC and sensor placement. Inlight of the present disclosure, one skilled in the art will appreciatethat either the flex-PCB technology or glass panel microfabricationtechnology used to create the MEA plate can be well suited for addingsensors and ICs. In one illustrative example, a 48-wellelectrophysiology culture plate comprises an EEPROM memory chip disposedon the MEA plate and within in a pocket formed on the biologic cultureplate. It is a further aspect of the present disclosure that the chipcan be used by the electronic data acquisition system to storeinformation about the electrophysiology culture plate such as, but notlimited to, the type of plate, electrode mapping, specific electrodeproperties and the like. In an additional or alternative aspect, thechip can be used to store user information about the experiment beingperformed such as, for example and without limitation, when theexperiment started, the types of cells cultured or the compounds,concentrations applied, and the like. In other aspects, the electronicspockets can also be used for other types of IC chips such as, forexample and without limitation, temperature sensors, CO₂ sensors,humidity sensors, pH sensors, O₂ sensors and the like.

In other aspects, the present disclosure can provide forelectrophysiology culture plates that can be sterilized using simpletreatments to eliminate the risk of cytotoxicity and do not requiresurface preparation (apart from standard biomolecular treatments) forcell culture applications.

In other aspects, the present disclosure provides for a culture wellplate configuration: operable to prevent communication or contaminationbetween adjacent wells. In a further aspect shown in FIG. 21, the lid2100 comprises a plurality of well caps 2102 configured to overlie eachof the culture wells. In another aspect, each of the plurality ofculture wells has the same height relative to the peripheral wall of theculture plate.

In other aspects shown in FIGS. 20A and 20B, the present disclosureprovides for culture well plates having culture wells configured toconcentrate the volume of the cells/biomolecular treatments depositedspecifically on the microelectrode area. In yet other aspects, thepresent disclosure provides for culture well plates having culture wellscomprising an upper diameter and a lower diameter, wherein the upperdiameter is greater than the lower diameter. In a further aspect, eachculture well can circumscribe either a conical or frustoconicalstructure on a lower portion of the well.

In another aspect shown in FIGS. 17A-19, the biologic culture plate andMEA plate contain at least one alignment feature 1701 configured todefine the directionality of the plate or align the high-throughputculture well plate to a die-cut adhesive and the electrode substrate orboth. In another aspect, once assembled, a keying feature 1702 can alignthe electrophysiology culture plate assembly to the docking mechanismand the high-density connectors located in the electronics unit.

Assembly and Packaging

In one aspect, the final assembly of the electrophysiology culture platecan be performed in a scalable fashion. Here, all of the steps ofassembly can accommodate assembly of large numbers of devicessimultaneously. As shown in FIG. 2, there are at least three componentsto join together: the 48-well MEA plate, a die-cut or laser-cut adhesiveand a biologic culture plate. In one illustrative example, the assemblyprocess can be performed on a 3.5 inch×5 inch fixture configured toassemble one device at a time. In a further aspect, the fixture can beconfigured to accommodate a plurality of devices. In a further example,the fixture can comprise at least 12 inch×18 inch panels. In anotheraspect, the adhesive can be precisely aligned under a stereomicroscopewith an accuracy of approximately 100 μm with the MEA plate and biologicculture plate in multiple steps of a vacuum-assisted compression bondingprocess. In an alternate aspect, compression can be applied to thenearly-final electrophysiology culture plate utilizing a laminationpress or a standard compression press.

Electronics Unit

In other aspects shown in FIGS. 22-23, the present disclosure canprovide for an electronics unit 2200 coupled to microelectrodes that canbe configured to stimulate the cell and record data therefrom,optionally, immediately thereafter. As one skilled in the art willappreciate, stimulation and recording immediately post-stimulation canbe complicated and is not readily available in current commercial MEAsystems because of the very different scales that stimulation andrecording occur on. In the specific case of neural tissues, hundreds ofmillivolts are required to achieve a response due to stimulation throughextracellular electrodes, while the same electrodes will show signals inthe tens of microvolts when the tissue or cell culture generates asignal. Thus, four orders of magnitude of disparity exist between thestimulation and recording signal and effects of this disparity on theelectrode renders signal recovery futile unless a recovery technique isused. This interference, commonly referred to as an artifact, includesthe saturation of the signal amplifying elements and its effects in thesignal processing chain during the recovery from these strong signals.

One aspect of the present disclosure provides for an electronics unitthat employs closed loop artifact suppression that can incorporatefeedback in the form of discharge amplifiers to quickly return theelectrode to a useful range, compensating for effects that traditional,open loop systems do not. In one aspect, artifact suppression can beimplemented by an ASIC configured to significantly reduce or eliminatethe stimulation artifact. Here, the ASIC can include an electrodeinterface, a path for stimulating the electrode, preamplifiers withbuilt in gain and bandwidth control, as well as multiplexing and outputbuffering. This custom design allows us to implement stimulation,artifact elimination, and recording on all 768 channels, which would notbe possible using commercial off the shelf parts. Such an electronicsunit is described in U.S. patent application Ser. No. 11/511,794 filedon Aug. 29, 2006, the contents of which are hereby incorporated byreference.

Alignment Features

In another aspect, the present disclosure can provide for at least onealignment feature integrated into the electrophysiology culture wellplate. In a further aspect, high-density electrical contacts can beconfigured to align to the electronic systems configured to impartfunctionality to the microelectrodes.

In another aspect, the present disclosure can provide fornon-restrictive electromechanical interfacing. More particularly, inorder to accommodate automated liquid handlers as well as experiments inwhich evaporation control is utilized, the present disclosure can allowfor addition or removal of the culture plate lid before or afterattachment of the apparatus to the electronics unit.

In other aspects, the present disclosure provides for electrophysiologyculture plates and an associated electronics unit having at least onepair of mating mechanical features to enable self-alignment between theelectrophysiology culture plate and the high-density connectorsassociated with the electronics unit. In yet other aspects, the presentdisclosure also provides for electrophysiology culture well plateshaving at least one pair of mating mechanical features configured tofacilitate alignment and attachment of the electrophysiology cultureplate in the electronic unit.

System Mechanics Features

In one aspect, vertical system integration within the SLAS footprint canbe achieved using high-density connectors within the electronic dataacquisition system configured to interface with the electrophysiologyculture plate through contact pads located on the bottom side of theelectrophysiology culture plate around at least a portion of a perimeterregion. In one aspect, such a system of high-density connectors does notrequire a connector on the MEA plate, thereby reducing the amount ofspace devoted to the connection interface. Such an interface can beimplemented through a pattern of contact pads that can be fabricatedthrough the standard flex-PCB processes or glass panel microfabricationprocesses, described above. In one aspect, the pins can be locatedaround the perimeter of the plate, and, in further aspects, the pins canbe located solely along the long sides in order to reduce interferencewith the culture wells. In light of the present disclosure, one skilledin the art will appreciate that such placement can be especiallyimportant for transparent substrates, where internal or backside tracesand pads within the well boundaries would interfere with opticalimaging. Additionally, the perimeter location of the pins andcorresponding contact pads can enable flexible well (e.g., 1-, 6-, 12-,24-, 48-, 96-, 384 and higher well count electrophysiology cultureplates) and electrode configurations without requiring changes such as,for example and without limitation, hardware, docking or connectivitychanges. In other aspects, the location of the contact pads in theperimeter region can also clear the backside for heater implementation.In one aspect, a heater can either be implemented in the electrodesubstrate PCB or be an external heater 2203 located beneath theelectrophysiology culture plate and configured to transfer heat to theculture wells through direct contact.

In another aspect, the perimeter contact pads can enable a dockingmechanism 2201 that can provide sufficient pressure to consistentlyengage the pogo-pins, without preventing topside access to the culturewells. The plastic high-throughput electrophysiology culture platedesign includes a reinforced bottom edge protrusion used to dock theplate in place. The MEA system uses a mechanism 2202 that grasps thisplate protrusion and applies force directly to these edges. Thisprevents the docking mechanism from interfering with topside well accessand lid placement/removal, when the plate can be engaged. The edgeprotrusion includes side tabs, making it compatible with automated cellculture and liquid dispensing equipment. With this configuration, thedocking mechanism places all of the docking force directly on the edgesof the plate, placing even pressure directly over the high-densityconnector interface.

Characterization Sterilization

PCB fabrication is typically not performed in a cleanroom environmentand can be prone to external contamination. Additionally material purityin PCB processes can be less stringent than traditional cleanroomprocesses. Toxicity from copper and nickel with potential leachingthrough the Kapton layer can be an additional concern. Lasermicromachining of Kapton is a process that can potentially releaseleachants on to the surface of the electrodes. Thus, in one aspect ofthe present disclosure, a sterilization process is provided to alleviateany issues with cytotoxicity across multiple cell lines (e.g., ratcortical, rat hippocampal, mouse cortical, stem cell, Doral RootGanglion (DRG) etc.) and multiple material surfaces (e.g., Kapton andSU-8).

In one aspect, sterilization of the electrophysiology culture platecomprises the use of deionized (DI) water, ethanol and heat to producehighly cytocompatible surfaces. In one aspect, the technique can involvecleaning the assembled HTMEAs in DI water (3 times) followed by anaseptic rinse in sterile 70% ethanol. Next, the device can be exposed to70% ethanol for 5 minutes in a passive setting and rinsed in 100%ethanol immediately following this step. The electrophysiology cultureplate can then be baked for 4-5 hours at 50° C. in a sterile oven andheat-sealed into plastic trays immediately after the oven bake.

Impedance Measurements

In another aspect of the present disclosure, electrical impedancespectroscopy of the fabricated electrophysiology culture plate can beused to evaluate the electrical properties of individual electrodes andcan provide feedback about processing steps, such as electroplatingissues or variations in the size of the microeletrodes indicating anissue with the laser micromachining process. Therefore, it can beimportant to establish the viability of each electrode before biologicaltesting. In one aspect, establishing electrode viability can beaccomplished with a Stanford Research SR785 (Stanford Research Systems,Sunnyvale, Calif.) two-channel dynamic signal analyzer augmented with acustom-built, controlled switching board that can allow for rapid,automated measurements of the magnitude and phase of microelectrodeimpedances across a large range of frequencies, e.g., 1 mHz to 100 kHz.In one illustrative example, impedance measurements can be performedbetween the microelectrode, a reference ground electrode and thecellular conducting media (Hank's Balanced Salt Solution), InvitrogenCorporation, Carlsbad, Calif.). Here, the fabricated and packaged48-well electrophysiology culture plate can be interfaced with thissetup and each electrode was scanned. FIG. 14 shows the impedancespectroscopy measurements of a representative set of microelectrodes outof the 768 that are distributed across the whole well. The electrodescan be well matched with an average impedance of about 70 to about 100kOhms at 1 kHz.

Noise Measurements

In other aspects, measurement of ambient noise of the microelectrodescan be useful for cell culture development and applications/assays thecell cultures can be used with. In light of the present disclosure, oneskilled in the art will appreciate that the baseline noise of anelectrode should be relatively low in order to record extracellularelectrophysiological activity from an electrogenic cell culture. In oneillustrative example, noise measurement was performed using the AxionMaestro System and the AxIS software. Here, the electrophysiologyculture plate was interfaced with the Maestro System and measurementswere made under minimum surrounding noise (e.g., no ambient lighting orblowers in hoods) between the microelectrodes, either ground orreference electrode integrated into each well, and the cellularconducting media. The baseline system noise with the nano-textured goldelectrodes was measured in saline and was found to be 4-5 μV RMS (200Hz-5 KHz), allowing for signals >10-15 μV to be consistently detected. Asnap shot of the data recorded from all the channels in theelectrophysiology culture plate is shown in FIG. 15, which illustratesthat the RMS noise of a single well in a 48-well MEA. Here, the RMSnoise lies in a range of from about 4 to about 5 microvolts ensuresneural signals having a value of less than about 10 to about 15microvolts can be consistently detected.

Cell Culture Protocols

In another aspect of the present disclosure, cell lines can be optimizedfor growth, survival and assay development on the electrophysiologyculture plates. Here, steps for cell culture protocol development cancomprise preparation of the media, preparation of the MEA surface andcoating biomolecular layers on the MEA surface before plating the cellson the electrophysiology culture plates. In order to illustrate thisaspect, development of rat and mouse cells lines on theelectrophysiology culture plates are described below. In a furtheraspect, forming a cell culture protocol as described herein can be thefirst step toward the creation of assays.

Rat Cortical Neurons

In one aspect, a cell culture protocol is provided for rat corticalneurons. Here, E18 rat cortical neurons can be obtained in a tissueformat (as a cortex pair) from commercial entities. First, the tissuecan be rinsed in HBSS, pre-warmed in dilute trypsin and then broken upinto a cellular suspension in a vortex. The cell suspension can then becentrifuged and individualized cells can be counted in a hemacytometerto get an accurate count of the total number of cells in suspension.

In another aspect, the media for cell growth (DMEM with glutamax andhorse serum in well-established concentrations and the surface of theelectrophysiology culture plates can be prepared separately. Theelectrophysiology culture plate is already sterilized as describedpreviously so that it can be ready for use in an experiment upon removalfrom the package. The electrophysiology culture plate can be removedinside a laminar hood after wiping the edges in ethanol.Polyethylenimine (PEI) can be coated on the top surface of the MEA andincubated for 1 hour. The PEI layer can be subsequently rinsed and theMEA can be air dried in a bio safety cabinet. Laminin (preparedseparately in pre-determined concentrations) can be coated on thesurface of the electrophysiology culture plates and incubated. As oneskilled in the art will appreciate in light of the present disclosure,the incubation integral to this step generally results in bettercellular adhesion to the MEA surface. Both these biomolecular treatmentscan be performed in a whole area or via dotting methods. In one aspect,the whole area technique can cover the entire well whereas, in analternate aspect, the dotting method accurately places both PEI andlaminin only on the electrode area as indicated in the schematic in toensure cell adhesion in the area of interest alone. One skilled in theart will appreciate the advantages and disadvantages to both techniques:The whole area method can be easier to implement but does incuradditional resources in terms of cells and coatings while the dottingmethod accurately places the cells in the area of interest but can beharder to implement manually in a high-throughput format. However, thedotting method can be well-suited for automated, robotic liquid handlersthat can accurately and efficiently deposit coatings/cells in ahigh-throughput format.

The cells can then be plated onto the MEA wells. In one aspect, theplating density depends on the application and can range from 2.5×10⁵cells/well and greater. The prepared media can be added to the wellsimmediately after the cells can be plated. The cells can be cultured inthe incubator with media changes every two or three days. Electricalactivity can be typically detectable in rat cortical cultures after 7days in-vitro (DIV).

Mouse Cortical Neurons

In another aspect, a mouse cortical protocol is provided that is similarto the rat cortical protocol described above with the followingmodifications: Here, cryopreserved primary neuronal cells dissociatedfrom rodent brains and spinal cords and, more particularly, E14/15embryonic C57 mouse cortical cell line from QBM, can be utilized forthese experiments. These cells can be preserved in cryo vials and,utilizing simple steps such as bringing the vial up to room temperatureand adding media with monitored mixing, these cells can be prepared forplating. Here, the media used for mouse cortical cell culture can be acombination of fetal bovine serum, L-glutamine, penicillin/streptomycinand B-27 supplement into Neurobasal.

The rest of the protocol is substantially similar to that described forrat cortical cells. In light of the present disclosure, one skilled inthe art will appreciate that cellular density and the biomoleculartreatments can be optimized for the mouse cells. Spontaneous activitycan be typically observable after 10 days in-vitro in the case of mousecells.

Cytotoxicity Evaluation

In another aspect, the cytocompatibility of the 48-well MEA (and all thematerials involved in the microfabrication and assembly of the MEA) canbe measured utilizing the CellTiter-Glo Luminescent Cell ViabilityAssay. This assay can be a homogeneous method for determining the numberof viable cells in culture based on the quantification of the AdinosineTri-Phosphate (ATP) present. ATP is regarded as a well-establishedindicator of metabolically active cells. The CellTiter-Glo® Assay can bedesigned for use with high-throughput formats, making it ideal forautomated high-throughput screening (HTS), cell proliferation andcytotoxicity assays. The homogeneous assay procedure can involve addingthe single reagent (CellTiter-Glo® Reagent) directly to cells culturedin serum-supplemented medium. Cell washing, removal of medium andmultiple pipetting steps are not required. The assay system is capableof detecting as few as 15 cells/well in a 384-well format in 10 minutesafter adding reagent and mixing.

In this aspect, the homogeneous “add-mix-measure” format can result incell lysis and generation of a luminescent signal proportional to theamount of ATP present. The amount of ATP can be directly proportional tothe number of cells present in culture. The CellTiter-Glo® Assaygenerates a “glow-type” luminescent signal, which has a half-lifegenerally greater than five hours, depending on cell type and mediumused. The extended half-life eliminates the need to use reagentinjectors and can provide flexibility for continuous or batch modeprocessing of multiple plates. The unique homogeneous format can avoiderrors that can be introduced by other ATP measurement methods thatrequire multiple steps.

In one illustrative example, cytotoxicity evaluation of the materialsinvolved in the fabrication of the 48-well electrophysiology cultureplate, a rat cortical neuronal culture as described supra was started onmultiple 48-well electrophysiology culture plates having variations inelectrode sizes in order to mimick potential exposure to leachants fromlaser micromachining, copper or FR-4; misaligned microelectrodes inorder to mimick similar issues; and normal well-aligned microelectrodes.Each of the 48-well electrophysiology culture plates were assembled andsterilized in the same manner. The effect of the potential cytotoxicitydue to the various factors listed above was evaluated with an ATP assayas described above. FIG. 16 depicts practically no difference betweenthe controls and the HTMEA wells with all the differing conditionsproving excellent cytocompatibility. There were minor differencesbetween the various 48 wells with the differing conditions. Additionallyvarious applications discussed below establish excellentcytocompatibility with multiple cell lines at various sites worldwide.

Exemplary Uses

In other aspects, the electrophysiology culture plates of the presentdisclosure can be utilized for a variety of high-throughput screeningapplications such as, for example and without limitation, diseasemodeling, phenotypic screening, toxicity testing and the like. Threeexemplary areas where high-throughput electrophysiology culture platesare becoming increasingly important are phenotypic screening, stem cellcharacterization, and toxicity/safety testing.

Phenotypic Screening

Between 1999 and 2008, despite a strong industry focus on target-basedscreening, the majority of first in class drugs were discovered throughphenotypic screening. This success is due, in part, to the fact thatdiscovery can be driven by desired effect, rather than an anticipatedtarget or mechanism of action. In drug safety applications, phenotypicin vitro screening of native cell types can provide a target-agnosticapproach, sensitive to acute effects that do not result in short termcytotoxicity. In neurons and other excitable cells, the most distinctivephenotype can be the action potential, and the transmission of thiselectrical excitation to neighboring cells.

The nature of this electrical signaling, however, presents technicalchallenges for established screening technologies. For example, changesin membrane voltage occur at speeds exceeding frame rates of highcontent imagers. Additionally, other instruments, such ashigh-throughput patch clamp, cannot collect measurements from multiplelocations within intact networks, and thus, do not address system-levelphenotypes. While neurons dissociated into in vitro cultures lack thecomplex organization of in vivo tissue, they do develop functionalnetworks that display correlated activity and chemical sensitivities,strongly representative of basic properties in vivo. Moreover, neurons,cardiomyocytes, and other cell types isolated from animal disease modelscan display network-level disease phenotypes in vitro.

Recently, the power of phenotypic screening has been amplified by theemergence of pluripotent stem (iPS) cells induced from adult humansomatic cells types. In fact, human neurons can now be differentiated insufficient quantities for high-throughput screening platforms. Among themost enticing possibilities for human excitable cells can be the abilityto screen compounds for effects on specific disease phenotypes.iPSC-derived neurons have been successfully characterized from manypatients, leading to a rapidly growing bank of disease models. Usinglow-throughput approaches, electrophysiological phenotypes have beenconfirmed for a subset of these, including ALS, Rett Syndrome, fragile Xsyndrome, and Timothy Syndrome. In other cases, morphological ormetabolic defects in patient-derived neurons strongly suggest thatelectrophysiological phenotypes can be observed in vitro.

Accordingly, one aspect of the present invention provides for an idealin vitro platform for phenotypic screening of excitable networkscomprising the following characteristics: (1) direct recording of thephenotypic signal of interest: voltage, (2) a signal resolution andsample rate sufficient to accurately capture action/field potentials,(3) numerous electrodes per well recorded simultaneously to assaysynaptic connectivity and action potential propagation, (4) label-free,non-invasive operation to avoid perturbation of natural cell function,and, (5) preservation of cellular interconnectivity. While severalcurrent screening technologies meet a subset of these criteria, only thehigh-throughput electrophysiology culture plate presents a scalablesolution that meets all criteria.

Stem Cell Characterization and Stem Cells as Research Tools

Stem Cell technology has accelerated rapidly in recent years.Differentiated excitable cells can be of interest for both therapy andresearch applications. Directing stem cell differentiation towards aspecific excitable cell type can be a complex process in which the finalcell type can take on undesired or unknown characteristics. As a result,such differentiated cells must be rigorously qualified. Many screeningtechnologies exist to examine the genetic and morphological features ofthese cells. However, only the high-throughput electrophysiology cultureplates can allow for the rapid characterization of thefunctional/electrophysiological behaviors of networks of excitablecells. Once such excitable differentiated cells can be characterized orqualified, they can be used as research tools (e.g., the phenotypicdisease models listed in the above section.)

Toxicity and Safety Testing

The National Academy of Sciences report on “Toxicity testing in the 21stcentury” highlighted the need for efficient in vitro methods to screenchemicals for their potential to cause toxicity. This report proposedthat high-throughput/high content in vitro screening (HTS/HCS) assayswould facilitate hazard identification for thousands of chemicals forwhich toxicological information can be lacking Such screening approacheswill need to link changes measured at the cellular or subcellular levelto adverse effects through a toxicity pathway so that there can beconfidence in predicting toxicological outcomes in vivo.

Many HTS/HCS endpoints assess changes in biochemical and/or cellularmorphology markers, such as enzyme activity, receptor binding affinity,cell morphology, or physiological endpoints, such as regulation ofintracellular calcium, sodium, membrane potential and ion channelfunction. When considering the problem of screening compounds for thepotential to disrupt nervous system function, physiological assessmentcan be crucial, as disruption of ion channels, receptors and otherimportant determinants of neuronal excitability can be key events in thetoxicity pathways of many known neurotoxicants. Disruption of neuronalexcitability produces substantial and rapid disruption of nervous systemphysiology, and often precedes or occurs in the absence of otherbiochemical or morphological changes. Examples include insecticides, avariety of convulsants and metals, as well as a wide range of naturaltoxins. However, current in vitro assays based on biochemical andmorphological changes are not optimized for detecting this type oftoxicity. These assays do not incorporate measurement of the key eventsin the toxicity pathways of such neurotoxicants (e.g. changes inintracellular ion concentrations can be secondary events), or are notamenable to collecting data at a rate that can capture the most rapidneurophysiological events, for example disruption of voltage-gatedsodium channels and action potential generation by pyrethroidinsecticides. By contrast, currently available electrophysiologicalapproaches are not well designed for toxicity screening, as these assaystypically consider only one potential target at a time (e.g. aparticular ion channel) and often employ non-neuronal expression systemsrather than neuronal tissue. Furthermore, neither biochemical nor HTSphysiological (e.g. patch clamp) approaches consider chemical effects onneuronal network function. Many of these traditional assays weredesigned as targeted screens for pharmaceutical-lead compounddevelopment and lack the ability to detect a broad spectrum of differentneurotoxicants. Thus, efficient screening assays that detect neurotoxicor neuroactive chemicals based on changes in function can be lacking,particularly those that can be sensitive to changes mediated bydisruption of a variety of different toxicity pathways.

One physiological approach that addresses these limitations can be invitro microelectrode array (MEA) recording. Using electrophysiologyculture plates, spontaneous and evoked activity in neuronal networks canbe recorded from a variety of different cell preparations, includingprimary cultures, tissue slices and intact retinas. Neuronal activity incultures grown on electrophysiology culture plates can be sensitive to avariety of drugs and chemicals and responds to a broad spectrum ofpharmaceutical compounds. As such, neuronal networks onelectrophysiology culture plates can be a potential method of assessingeffects of many different pharmacological classes of drugs and chemicalson nervous system function. To date, assessment of chemical effectsusing electrophysiology culture plates has been primarily on achemical-by-chemical basis to understand the toxicity of individualchemicals or chemical classes Use of electrophysiology culture plateshas been proposed as an in vitro neurotoxicity screening method and arecent study demonstrated consistent reproducibility and reliability ofMEA measurements across five laboratories. One limitation of traditionalMEA approaches can be that throughput of this methodology has been lowand can be limited by the MEA plates and hardware. The discloseddisclosure overcomes such limitation to allow for comprehensive safetytesting at high-throughputs.

Accordingly, FIGS. 1-23, and the corresponding text, provide a number ofdifferent devices, systems, methods and mechanisms for high-throughputelectrophysiology. In addition to the foregoing, implementationsdescribed herein can also be described in terms, acts, and steps in amethod for accomplishing a particular result. For example, a methodcomprising plating, stimulating and recording data from a cell cultureis described concurrently above with reference to the components anddiagrams of FIGS. 1-23.

The present invention can thus be embodied in other specific formswithout departing from its spirit or essential characteristics. Thedescribed aspects are to be considered in all respects only asillustrative and not restrictive. The scope of the invention is,therefore, indicated by the appended claims rather than by the foregoingdescription. All changes that come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

We claim:
 1. An electrophysiology culture plate comprising: atransparent MEA plate having: a substrate having a plurality of viasextending from an upper surface to a lower surface and each via being inelectrical contact with each of a plurality of contact pads disposed onthe lower surface, a first layer disposed on the upper surface of thesubstrate comprising a plurality of MEA arrays in electricalcommunication with at least a first routing layer wherein each MEA arraycomprises a plurality of reference electrodes and a plurality ofmicroelectrodes and wherein the first routing layer is in electricalcommunication with a select number of the plurality of vias, and a firstinsulating layer disposed on the first layer; and a biologic cultureplate having a plurality of culture wells, wherein each culture welldefines an interior cavity having a bottom surface that is at leastpartially transparent and is positioned in registration with a selectoptical port; wherein the MEA plate underlies and is coupled to thebiologic culture well plate such that each MEA array is operativelycoupled to one culture well of the plurality of culture wells; whereineach microelectrode and each reference electrode are in electricalcommunication with the interior cavity of a select culture well of theplurality of culture wells through the bottom surface of the selectculture well.
 2. The electrophysiology culture plate of claim 1, furthercomprising: a second layer disposed on the lower surface of thesubstrate comprising at least a second routing layer and wherein thesecond routing layer is in electrical communication with a select numberof the plurality of vias, and a second insulating layer disposed on thesecond layer.
 3. The electrophysiology culture plate of claim 1, whereinthe plurality of reference electrodes comprises two referenceelectrodes.
 4. The electrophysiology culture plate of claim 3, whereinat least one of the two reference electrodes comprises two referenceelectrodes connected in series.
 5. The electrophysiology culture plateof claim 1, wherein the biologic culture plate is bonded to thesubstrate.
 6. The electrophysiology culture plate of claim 1, whereinthe microelectrodes are nanotextured.
 7. The electrophysiology cultureplate of claim 1, wherein the microelectrodes are nanoporous.
 8. Theelectrophysiology culture plate of claim 1, wherein each culture wellfurther comprises an upper diameter and a lower diameter and wherein thelower diameter is less than the upper diameter.
 9. The electrophysiologyculture plate of claim 1, wherein the electrophysiology culture platefurther comprises a lid configured to be selectively coupled to aperipheral edge of the biologic culture plate.
 10. The electrophysiologyculture plate of claim 9, wherein the lid further comprises a peripheralwall and wherein the peripheral wall is double baffled.
 11. Theelectrophysiology culture plate of claim 9, wherein the lid comprises anindividual well cap for each culture well.
 12. The electrophysiologyculture plate of claim 1, wherein the bottom side contact pads arepositioned substantially on a peripheral region of the monolithic MEAplate.
 13. The electrophysiology culture plate of claim 1, wherein theculture plate is configured to prevent communication of the contents ofeach culture well of the plurality of culture wells disposed thereon.14. The electrophysiology culture plate of claim 1, wherein at least oneof a length, a width, and a thickness of the electrophysiology cultureplate conforms to at least one of American National Standards Institutestandards and Society for Lab Automation and Screening standards. 15.The electrophysiology culture plate of claim 1, wherein theelectrophysiology culture plate has a length of between about 127.13 toabout 127.83 mm.
 16. The electrophysiology culture plate of claim 1,wherein the electrophysiology culture plate has a width of between about85.23 to about 85.73 mm.
 17. The electrophysiology culture plate ofclaim 1, wherein the electrophysiology culture plate has a thickness ofbetween about 14.1 to about 14.6 mm.
 18. A system comprising: anelectrophysiology culture plate comprising: a transparent monolithic MEAplate having: a substrate having a plurality of vias extending from anupper surface to a lower surface and each via being in electricalcontact with each of a plurality of contact pads disposed on the lowersurface, a first layer disposed on the upper surface of the substratecomprising a plurality of MEA arrays in electrical communication with atleast first routing layer wherein each MEA array comprises a pluralityof reference electrode and a plurality of microelectrodes and whereinthe first routing layer is in electrical communication with a selectnumber of the plurality of vias., and a first insulating layer disposedon the first layer; and a biologic culture plate having a plurality ofculture wells, wherein each culture well defines an interior cavityhaving a bottom surface that is at least partially transparent and ispositioned in registration with a select optical port; wherein the MEAplate underlies and is coupled to the biologic culture well plate suchthat each MEA array is operatively coupled to one culture well of theplurality of culture wells; wherein each microelectrode and eachreference electrode are in electrical communication with the interiorcavity of a select culture well of the plurality of culture wellsthrough the bottom surface of the select culture well; and an electronicunit configured to stimulate and record data from at least onemicroelectrode of a select MEA array, wherein the electronic unitcomprises: a receiving cavity having an interior bottom surfaceconfigured to receive an electrophysiology culture plate, a high-densityconnector array disposed on the interior bottom surface of the receivingcavity configured to establish electrical contact between at least onemicroelectrode of the plurality of microelectrodes and the electronicunit, and a means of exerting a predetermined force to maintainelectrical contact between the contact pads disposed on theelectrophysiology culture plate and the high-density connector array.19. The system of claim 18, wherein the electrophysiology culture platefurther comprises: a second layer disposed on the lower surface of thesubstrate comprising at least a second routing layer and wherein thesecond routing layer is in electrical communication with a select numberof the plurality of vias, and a second insulating layer disposed on thesecond layer.
 20. The system of claim 18, wherein the receiving cavitycomprises at least one positioning pin extending from the bottom surfaceand the culture plate further comprises at least one positioning holeconfigured to receive the corresponding positioning pin.
 21. Theelectrophysiology culture plate of claim 18, wherein theelectrophysiology culture plate further comprises a lid configured to beselectively coupled to a peripheral edge of the biologic culture plate.22. The electrophysiology culture plate of claim 21, wherein the lidfurther comprises a peripheral wall and wherein the peripheral wall isdouble baffled.
 23. The electrophysiology culture plate of claim 21,wherein the lid comprises an individual well cap for each culture well.24. The system of claim 21, wherein the lid is configured to beselectively removed when the culture plate is secured in the electronicsunit.
 25. The system of claim 18, wherein the culture plate well isconfigured to self-align with the high-density connector array.
 26. Thesystem of claim 18, wherein the culture plate is configured to preventcommunication of the contents of each culture well of the plurality ofculture wells disposed thereon.
 27. The system of claim 18, a means ofexerting a predetermined force to maintain electrical contact betweenthe contact pads disposed on the electrophysiology culture plate and thehigh-density connector array comprises a lever.
 28. The system of claim27, wherein the lever is configured to transmit pressure to a clampconfigured to receive the electrophysiology culture plate.
 29. Thesystem of claim 18, wherein the electronic unit is configured tosimultaneously stimulate and record date from at least onemicroelectrode of a select MEA array.
 30. The system of claim 18,further comprising a heating element disposed on the interior bottomsurface of the receiving cavity of the electronic unit.